Vascular Drug Research Center
The Vascular Drug Research Center has been formed in September 2008 as a result of close collaborations, which have been developing between the labs of participating PIs over recent years.
The mission of the Center is to discover and develop novel therapeutics that will advance drug therapy in a spectrum of vascular diseases. The Center will foster research to characterize new drug targets based on understanding of both receptor- and transport mechanisms and of metabolic pathways.
By encouraging collaboration with colleagues both inside and outside of the Department of Pharmaceutical Sciences, the Center for Vascular Drug Research will raise the research profile of the School of Pharmacy, enhance the competitiveness of Center members for extramural funding and increase the attractiveness of TTUHSC SOP for prospective faculty members.
Members of the Center and Their Research Interests
All current members are faculty in the Department of Pharmaceutical Sciences, TTUHSC School of Pharmacy. A brief overview of research interests and methods used in the individual labs follows:
Dr. Abbruscato’s group investigates the role of the glucose carrier GLUT1 and of the Sodium Dependent Glucose Transporter (SGLT) at the blood-brain barrier (BBB). In particular, they focus on the regulation of SGLT under pathophysiological conditions such as ischemia/reperfusion injury (stroke). Selective pharmacological modulation of SGLT could have clinical implications: (i) inhibition of SGLT at the BBB could be beneficial in stroke by limiting excessive glucose delivery to brain and brain edema (because SGLT transfers 210 molecules of water for each Na+ ion and glucose molecule); (ii) enhancing SGLT activity in neurons could be protective by reducing oxidative stress; (iii) SGLT is involved in tight junction integrity and its induction in BBB endothelial cells could help to maintain brain vascular integrity during ischemia and prevent development of vasogenic edema. Further studies on the expression and regulation of SGLT in vivo with temporary focal ischemia combined with reperfusion would lead to a better understanding as to how it can be utilized as a therapeutic target in stroke and other CNS disorders.
Based on substantial work in his lab and by others, he believes that the Na+, K+, 2Cl- Cotransporter (NKCC) plays a critical role in cellular edema associated with stroke. NKCC protein expression and function has been proven to be modulated by PKC in ischemia in our lab and could provide a promising drug target. One exciting avenue to pursue is the “cross-talk” between NKCC and opioid receptors. Classically, opioid receptor activation stimulates K+ channels and suppresses Ca2+ current into the cytoplasm of neurons. Opioid receptor activation during stoke has been shown to modulate other key ion channels by the activation of protein kinase C (PKC). Preliminary experiments in Dr. Abbruscato’s lab have shown that the selective δ agonist, DPDPE, μ agonist, DAMGO and δ/μ receptor agonist, biphalin, significantly decreased water gain induced in hippocampus slices subjected to oxygen-glucose deprivation. All the anti-edematous effects of opioid agonists are reversed by naloxone. Future experiments will attempt to decipher the contribution of NKCC at the neurovascular unit as a possible target for the anti-edematous effects of opioids.
The spectrum of methods in Dr. Abbruscato’s lab includes the following in vitro techniques:
Models of the BBB (bovine primary, mouse cell line); hypoxia chamber for reduced oxygen/glucose experiments; trans-endothelial cell resistance measurement; mouse primary neuronal and astrocytic cell culture; hippocampal brain slicing and oxygen glucose deprivation experiments; Western blot analysis. Available in vivo techniques include permanent and reversible mouse MCAO; infarct and edema ratio; locomotor testing for neurologic outcome post ischemia; Blood-CNS pharmacokinetics; mouse or rat brain endothelial cell isolation.
Due to the presence of the blood-brain barrier, the development of sophisticated delivery systems is required in order to exploit the therapeutic potential of macromolecular drugs (e.g., growth factors, antibodies, DNA or RNA-based drugs) for brain diseases. This is also true in stroke, because the BBB remains physically intact for several hours after onset of ischemia. Dr. Bickel’s group investigates the use of receptor-mediated uptake mechanisms at the BBB for drug targeting and delivery. His lab performed preliminary studies with the prototype target at the BBB, the transferrin receptor, and showed that this endothelial receptor maintains a level of binding/transport activity during ischemia/reperfusion injury, which appears sufficient for drug targeting. These data explain the success of delivery of growth factors via transferrin receptors by other labs. Recently Dr. Bickel’s lab completed studies in models of transient and permanent brain ischemia in mice, which showed significant therapeutic effect of anti-inflammatory oligonucleotide drugs when delivered to BBB transferrin receptors. The next steps are to characterize at the cellular level, where the pharmacological effect seen in the whole animal models actually occur (e.g., activity on endothelial cells, neurons, and/or glial cells?).
Another focus of research is the effort to find and characterize BBB-specific targets for drug delivery. Receptor systems like the transferrin receptor or insulin receptor are highly expressed at the BBB and have been successfully used in preclinical studies for brain targeting, but these receptors are obviously not brain-specific. A vascular targeting strategy based on receptors only expressed at the BBB has thus far been elusive. This is an attractive goal, because it may reduce the risk of unwanted effects in peripheral organs, and potentially allow regional targeting to brain tissue affected by disease (e.g., ischemic brain tissue).
In addition to the middle cerebral artery occlusion (MCAO) stroke model, brain microdialysis has been recently established in Dr. Bickel’s lab in collaboration with Dr. Jochen Klein (now at the College of Pharmacy at University of Frankfurt, Germany). The combination of these techniques provides a unique opportunity to monitor pharmacological effects in vivo under stroke conditions.
The following methods are routinely used in Dr. Bickel’s group:
In vitro techniques:
Brain endothelial cell culture (cell lines from human and murine origin); production and purification of monoclonal antibodies from hybridoma supernatant; transport studies with endothelial cell monolayers (Transwell); cell adhesion assays ; flowcytometry ; radiotracer labeling; radioligand binding assays; ELISA; Northern blotting; Real time PCR; gel shift assays; HPLC and FPLC for analytical and semipreparative purpose; protein conjugations; supravital staining of brain slices (TTC); immunohistochemistry; wide field fluorescence microscopy and confocal microscopy.
Ex vivo methods:
Autoradiography of brain cryostat sections following administration of radioactive tracers in vivo; capillary depletion technique to characterize brain uptake; brain capillary and endothelial cell isolation.
In vivo techniques:
Middle Cerebral Artery Occlusion (MCAO) by intraluminal suture for induction of permanent or transient brain ischemia in mice; brain microdialysis; Laser Doppler Flowmetry; carotid artery catheterization in mice (and rats) for arterial blood sampling in pharmacokinetic studies; carotid artery catheterization for vascular brain perfusion; sensorimotor testing in mice (Corner test).
Dr. Mehvar’s research is focused on the pharmacological approaches to reduce ischemia-reperfusion (IR) injury in the liver. Hepatic ischemia-reperfusion injury is a serious, yet so far unavoidable, complication of many clinical situations such as liver transplantation, major liver resection surgery (Pringle maneuver), and hemorrhagic and septic shock. Although all of the in vivo conditions resulting in the interruption of blood supply to the liver produce warm IR injury, liver transplantation is unique in that, in addition to a variable degree of warm IR injury during organ retrieval and/or transplantation, it is also subjected to cold (storage) IR injury during organ preservation. Initially, we focused on the pharmacokinetic-based delivery approaches to target anti-inflammatory drugs to the liver for prevention of IR injury. Our current emphasis, however, is on the role of cytochrome P450 in generation of reactive oxygen species (ROS) and pharmacological approaches to inhibit P450 generation of ROS.
Part of this project is the live-cell imaging of hypoxia/reoxygenation injury in hepatocytes by confocal microscopy, which recently stimulated collaboration with Dr. Bickel’s lab.
Major techniques used in our laboratory are: in vivo warm hepatic IR model in rats; ex vivo warm hepatic IR model in rats; ex vivo cold hepatic IR model in rats; rat liver transplantation model; macromolecular-based drug delivery to the liver; primary cultures of rat hepatocytes; P450 content and activity assays; HPLC analysis of small molecules and macromolecules in biological samples; pharmacokinetic dosing and sampling methods in rodents; and pharmacokinetic and drug metabolism data analysis.
With the collaboration of Dr. Ulrich Bickel (TTUHSC) and Dr. George Gokel (University of Missouri, St. Louis), I recently submitted an RO1 proposal to synthesize and test a series of compounds that are predicted to increase eNOS activity. Our preliminary studies showed that triacsin C, the lead compound in the series, inhibits long chain fatty acyl CoA synthetase, inhibits eNOS palmitoylation, increases NO production in both cultured endothelial cells and intact aorta, and enhances methacholine induced relaxation of vascular smooth muscle. It is anticipated that these compounds may have clinical utility in treatment of hypertension. There is preliminary evidence that triacsin C and like compounds may have neuroprotective effects in an experimental model of stroke. In both a temporary (1 hour of occlusion followed by 24 hours reperfusion) and permanent (24 hours occlusion) stroke model, triacsin C significantly reduced the cerebral infarct volume. Other data indicates that triacsin C may not only increase eNOS activity, it may also inhibit iNOS expression. Hypertension is the single most important risk factor for stroke. It is estimated that about 80% of stroke victims are hypertensive. Our observations with triacsin C suggest that it is possible to develop an antihypertensive drug that would also have the benefit of reducing neurologic damage in the event of a stroke. The Ischemia/Reperfusion Injury Center would provide an important mechanism for feedback and discussion of this research.
Major Biochemical Techniques used in my laboratory include assay of catalytic activities of long chain fatty acyl CoA synthetase and nitric oxide synthase, radioimmunoassay of prostaglandins, Griess reaction, and extraction and analysis of total tissue/cellular lipid. We also do cell culture (human and rat coronary endothelial cells), endothelial shear stress measurement, western blotting, immunoprecipitation, and siRNA. In addition, I have extensive experience with and am fully equipped to do the Langendorf perfused heart preparation.